zFGF5-stimulated uptake may be evaluated, for example, in an assay for insulin-stimulated glucose transport. Primary adipocytes or NIH 3T3 L1 cells (ATCC No. CCL-92.1) are placed in GIBCO® DMEM (Invitrogen, Carlsbad, Calif.) containing 1 g/l glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine. After two to five hours of culture, the medium is replaced with fresh, glucose-free GIBCO® DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine. Appropriate concentrations of zFGF5, insulin or IGF-1, or a dilution series of the test substance, are added, and the cells are incubated for 20-30 minutes. 3H or 14C-labeled deoxyglucose is added to ≈50 μM final concentration, and the cells are incubated for approximately 10-30 minutes. The cells are then quickly rinsed with cold buffer (e.g. PBS), then lysed with a suitable lysing agent (e.g. 1% SDS or 1 N NaOH). The cell lysate is then evaluated by counting in a scintillation counter. Cell-associated radioactivity is taken as a measure of glucose transport after subtracting non-specific binding as determined by incubating cells in the presence of cytochalasin b, an injibitor of glucose transport. Other methods include those described by, for example, Manchester et al., Am. J. Physiol. 266(Enndocrinol. Metab. 29):E326-E333, 1994 (insulin-stimulated glucose transport).
Although administration of zFGF5 alone is sufficient to provide the delivery of the chondrogenic peptides of the present method, there may be clinical situations where additional drugs are combined in the admixture. Examples of other drugs which may be clinically indicated include anti-inflammatory drugs such as nonspecific and specific cyclooxygenase-2 inhibitors, non-steriodal and steroidal anti-inflammatory drugs. Some of the nonspecific COX inhibitors that could be used in the present invention include salicylic acid and derivatives, such as aspirin or sulfasalazine, para-aminophenol derivatives, such as acetaminophen, indole and indene acetic acids, such as indomethacin or sulindac, arylprpionic acids, such as ibuprofen, naproxen, or oxaprozin, anthranilic acids, such as mefenamic acid, enolic acids including oxicams, or alkanonoes, such as nabumentone. Specific COX-2 inhibitors would be diaryl-substituted fuanonoes (Refecoxib), diaryl-substituted pyrazoles (Celecoxib), indole acetic acids (Etodolac) and sulfonaildes (Nimesulide). Additionally, steroids, such as dexamethazone, prednisone, triamcinolone, or methylprednisone, are among the drugs that could be used. Other types of drugs suitable for the present invention would be inhibitors of the tumor necrosis factor family, such as ENBREL® or TACI-Ig, IL-1 antagonists such as KINERET®, antagonists of IL-18 and IL-15, and immunosuppressive drugs such as cyclosporine. In addition, zFGF5 may be administered with inhibitors of the CC (MCP-1, RANTES, MIP-1alpha, and MIP-1beta) and CXC (IL-8 and GRO-alpha) chemokine family.
Mitogenic activity is assayed by measurement of 3H-thymidine incorporation based on the method of Raines and Ross (Meth. Enzymology 109:749-773, 1985). Briefly, quiescent cells are plated cells at a density of 3×104 cells/ml in an appropriate medium. A typical growth medium is Dulbecco's Growth Medium (GIBCO-BRL, Gaithersburg, Md.) containing 10% fetal calf serum (FCS). The cells are cultured in 96-well plates and allowed to grow for 3-4 days. The growth medium is removed, and 180 μl of DFC (Table 5) containing 0.1% FCS is added per well. Half the wells have zFGF5 protein added to them and the other half are a negative control, without zFGF5. The cells are incubated for up to 3 days at 37° C. in 5% CO2, and the medium is removed. One hundred microliters of DFC containing 0.1% FCS and 2 μCi/ml 3H-thymidine is added to each well, and the plates are incubated an additional 1-24 hours at 37° C. . The medium is aspirated off, and 150 μl of trypsin is added to each well. The plates are incubated at 37° C. until the cells detached (at least 10 minutes). The detached cells are harvested onto filters using an LKB Wallac 1295-001 Cell Harvester (LKB Wallac, Pharmacia, Gaithersburg, Md). The filters are dried by heating in a microwave oven for 10 minutes and counted in an LKB BETAPLATE™ 1250 scintillation counter (LKB Wallac) as described by the supplier.
For the mapping of zFGF5 with the “GeneBridge 4 RH Panel”, 25 μl reactions were set up in a 96-well microtiter plate (Stratagene, La Jolla, Calif.) and used for PCR in a RoboCycler Gradient 96 thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2.5 μl 50X “ADVANTAGE® KlenTaq Polymerase Mix” (Clontech), 2 μl dNTPs mix (2.5 mM each; Perkin-Elmer, Foster City, Calif.), 1.25 μl sense primer, ZC11677 (SEQ ID NO: 4) 1.25 μl antisense primer, ZC12053 (SEQ ID NO: 5).
2.5 μl “REDILOAD™” (Research Genetics, Inc), 0.5 μl “ADVANTAGE® KlenTaq Polymerase Mix” (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH2O for a total volume of 25 μl. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle of 4 minutes at 94° C., 35 cycles of 1 minute at 94° C., 1.5 minute annealing at 66° C. and 1.5 minute extension at 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C. The reactions were separated by electrophoresis on a 3% NUSIEVE® GTG agarose gel (FMC Bioproducts, Rockland, ME.).
A total of 39 adult female goats were used and were divided into thirteen groups of three goats each. A full thickness cartilage lesion (6.25 mm wide×2 5 mm deep) was created in the distal femoral trochlear sulcus of each goat. zFGF5 (0, 0.04, 0.4, 4.0 or 40.0 ug) was delivered directly into the defects either alone or suspended in a bio-degradable fast release (degradation over 1-2 weeks) or slow release (degradation over 2-4 weeks) poly(lactide-co-glycolide) matrix that solidified in situ (ATRIGEL®, QLT Inc., Vancouver, Calif.). Eight weeks after treatment, the defect sites were scored for gross morphology and harvested. Sections were taken through the center of the lesions and repair of subcondral bone was evaluated by contact radiography and by staining of adjacent sections with H&E. Formation of chondral tissue was evaluated by staining of adjacent sections with safranin-O. Degeneration of adjacent articular cartilage was assessed microscopically as decreased chondrocyte cell density and loss of safranin-O staining within cartilage adjacent to the lesions. Sections were scored in two ways: initially they were scored in a blinded fashion using the a semi-quantitative scoring scale (Frenkel SR et al., J Bone Joint Surg Br 1997, 79: 831-6); this was followed with an unblinded qualitative comparative analysis of slides from each group.
FGF-8 has five exons, in contrast to the other known FGFs, which have only three exons. The first three exons of FGF-8 correspond to the first exon of the other FGFs (MacArthur et al., Development 121:3603-3613, 1995.) The human gene for FGF-8 codes for four isoforms which differ in their N-terminal regions: FGF isoforms a, b, e, and f; in contrast to the murine gene which gives rise to eight FGF-8 isoforms (Crossley et al., 1995, ibid.) Human FGF-8a and FGF-8b have 100% homology to the murine proteins, and FGF-8e and FGF-8f proteins are 98% homologous between human and mouse (Gemel et al., Genomics 35:253-257, 1996.)
Heart disease is the major cause of death in the United States, accounting for up to 30% of all deaths. Myocardial infarction (MI) accounts for 750,000 hospital admissions per year in the U.S., with more than 5 million people diagnosed with coronary disease. Risk factors for MI include diabetes mellitus, hypertension, truncal obesity, smoking, high levels of low density lipoprotein in the plasma or genetic predisposition.
Cardiac hyperplasia is an increase in cardiac myocyte proliferation, and has been demonstrated to occur with normal aging in the human and rat (Olivetti et al., J. Am. Coll. Cardiol. 24(1):140-9, 1994 and Anversa et al., Circ. Res. 67:871-885, 1990), and in catecholamine-induced cardiomyopathy in rats (Deisher et al., Am. J. Cardiovasc. Pathol. 5(1):79-88, 1994.) Whether the increase in myocytes originate with some progenitor cell, or are a result of proliferation of a more terminally differentiated cell type, remains controversial.
However, because infarction and other causes of myocardial necrosis appear to be irreparable, it appears that the normal mechanisms of cardiac hyperplasia cannot compensate for extensive myocyte death, and there remains a need for exogenous factors that promote hyperplasia and ultimately result in renewal of the heart's ability to function.
Stroke is caused by either cerebral thrombosis, embolism, or subarachnoid or cerebral hemorrhage, and results in ischemia in approximately 80% of occurrences. Stroke is a major health problem disabling over three million people in the United States, with 550,0000 Americans suffering stroke each year, of which 150,000 of those affected will die. The current treatments to prevent tissue damage resulting from stroke are very limited and require administration within an hour of onset of the stroke. While there are more drugs available to try to prevent reoccurrence of stroke, they are not without some serious drawbacks, including the development of intracranial hemorrhaging, gastrointestinal bleeding and neutropenia. Therefore, any therapeutics that promote angiogenesis, promote neurite outgrowth, or survival of neurons in necrotic areas of the central nervous system with some specificity will be valuable. The molecules of the present invention have been shown to promote growth in specific tissues, including neuronal tissue.
Bone remodeling is the dynamic process by which tissue mass and skeletal architecture are maintained. The process is a balance between bone resorption and bone formation, with two cell types thought to be the major players. These cells are the osteoblast and osteoclast. Osteoblasts synthesize and deposit matrix to become new bone. The activities of osteoblasts and osteoclasts are regulated by many factors, systemic and local, including growth factors.
While the interaction between local and systemic factors has not been completely elucidated, there does appear to be consensus that growth factors play a key role in the regulation of both normal skeletal remodeling and fracture repair. Some of the growth factors that have been identified in bone include: IGF-I, IGF-II, TGF-β1, TGF-β2, bFGF, aFGF, PDGF and the family of bone morphogenic proteins (Baylink et al., J. Bone Mineral Res. 8 (Supp. 2):S565-S572, 1993).
When bone resorption exceeds bone formation, a net loss in bone results, and the propensity for fractures is increased. Decreased bone formation is associated with aging and certain pathological states. In the U.S. alone, there are approximately 1.5 million fractures annually that are attributed to osteoporosis. The impact of these fractures on the quality of the patient's life is immense. Associated costs to the health care system in the U.S. are estimated to be $5-$10 billion annually, excluding long-term care costs.
Other therapeutic applications for growth factors influencing bone remodeling include, for example, the treatment of injuries which require the proliferation of osteoblasts to heal, such as fractures, as well as stimulation of mesenchymal cell proliferation and the synthesis of intramembraneous bone which have been indicated as aspects of fracture repair (Joyce et al. 36th Annual Meeting, Orthopaedic Research Society, Feb. 5-8, 1990. New Orleans, La.).
Replacement of damaged articular cartilage caused either by injury or defect is a major challenge for physicians, and available treatments are considered unpredictable and effective for only a limited time. Therefore, the majority of younger patients either do not seek treatment or are counseled to postpone treatment for long as possible. When treatment is required, the standard procedure is a total joint replacement or penetration of the subchondral bone to stimulate fibrocartilage deposition by chondrocytes. While deposition of fibrocartilage is not a functional equivalent of articular cartilage, it is at the present the best available treatment because there has been little success in replacing articular cartilage. Two approaches to stimulating deposition of articular cartilage that are being investigated are: stimulating chondrocyte activity in vivo and ex vivo expansion of chondrocytes and their progenitors for transplantation (Jackson et al., Arthroscopy: The J. of Arthroscopic and Related Surg. 12:732-738, 1996). In addition, regeneration or repair of elastic cartilage is valuable for treating injuries and defects to ear and nose. Any growth factor with specificity for chondrocytes lineage cells that stimulates those cells to growth, differentiate or induce cartilage production would be valuable for maintaining, repairing or replacing articular cartilage.
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.